Inhibitory phenotype of HBV-specific CD4+ T-cells is characterized by high PD-1 expression but absent coregulation of multiple inhibitory molecules.

BACKGROUND: T-cell exhaustion seems to play a critical role in CD8+ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4+ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4+ T-cell failure. METHODS: The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4+ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4+ T-cell proliferation and cytokine production. RESULTS: CD4+ T-cell responses during chronic HBV infection was characterized by reduced Tetramer+CD4+ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4+ T-cell expansion almost in treated patients with viral control. CONCLUSION: HBV-specific CD4+ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4+ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4+ T-cell functionality with heterogeneous patterns of CD4+ T-cell rejunivation.

Lack of variant specific CD8+ T-cell response against mutant and pre-existing variants leads to outgrowth of particular clones in acute hepatitis C.

BACKGROUND: CTL escape mutations have been described during acute hepatitis C in patients who developed chronic disease later on. Our aim was to investigate the mutual relationship between HCV specific CD8+ T cells and evolution of the viral sequence during early acute HCV infection. RESULTS: We sequenced multiple clones of NS3 1406 epitope in 4 HLA-A*02 patients with acute hepatitis C genotype 1b infection. Pentamers specific for the variants were used to monitor the corresponding CD8+ T cell response. We observed outgrowth of mutations, which induced only a weak and thus potentially insufficient CD8+ T cell response. In one patient we observed outgrowth of variant epitopes with similarities to a different genotype rather than de novo mutations most probably due to a lack of responsiveness to these likely pre-existing variants. We could show that in acute hepatitis C CTL escape mutations occur much earlier than demonstrated in previous studies. CONCLUSIONS: The adaption of the virus to a new host is characterized by a high and rapid variability in epitopes under CD8+ T cell immune pressure. This adaption takes place during the very early phase of acute infection and strikingly some sequences were reduced below the limit of detection at some time points but were detected at high frequency again at later time points. Independent of the observed variability, HCV-specific CD8+ T cell responses decline and no adaption to different or new antigens during the course of infection could be detected.

Inhibitory molecules that regulate expansion and restoration of HCV-specific CD4+ T cells in patients with chronic infection.

BACKGROUND & AIMS: Inhibitory receptors such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen (CTLA)-4 mediate CD8+ T-cell exhaustion during chronic viral infection, but little is known about roles in dysfunction of CD4+ T cells. METHODS: We investigated the functions of inhibitory molecules on hepatitis C virus (HCV)-, influenza-, and Epstein-Barr virus (EBV)-specific CD4+ T cells in patients with chronic infections compared with patients with resolved HCV infection and healthy donors. Expression of PD-1, CTLA-4, CD305, and CD200R were analyzed on HCV-specific CD4+ T cells, isolated from peripheral blood using major histocompatibility complex class II tetramers. We investigated the effects of in vitro inhibition of various inhibitory pathways on proliferation and cytokine production by CD4+ T cells, and we compared these effects with those from inhibition of interleukin (IL)-10 and transforming growth factor (TGF)-ß1. RESULTS: PD-1 and CTLA-4 were up-regulated on virus-specific CD4+ T cells from patients with chronic HCV infections. PD-1 expression was lower on influenza- than on HCV-specific CD4+ T cells from subjects with chronic HCV infection, whereas CTLA-4 was expressed at similar levels, independent of their specificity. CD305 and CD200R were up-regulated in HCV resolvers. Blockade of PD-L1/2, IL-10, and TGF-ß1 increased expansion of CD4+ T cells in patients with chronic HCV, whereas inhibition of IL-10 and TGF-ß1 was most effective in restoring HCV-specific production of interferon gamma, IL-2, and tumor necrosis factor α. CONCLUSIONS: We characterized expression of inhibitory molecules on HCV-, influenza-, and EBV-specific CD4+ T cells and the effects of in vitro blockade on CD4+ T-cell expansion and cytokine production. Inhibition of PD-1, IL-10, and TGF-ß1 is most efficient in restoration of HCV-specific CD4+ T cells.

Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity.

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function.

UNLABELLED: Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic hepatitis B virus (HBV) infection. Yet few receptors have been characterized in detail and little is known about their complex regulation. In the present study, we investigated the role of the signaling lymphocyte activation molecule (SLAM)-related receptor CD244 and of programmed death 1 (PD-1) in HBV infection in 15 acutely and 66 chronically infected patients as well as 9 resolvers and 21 healthy controls. The expression of CD244, PD-1, and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) was analyzed in virus-specific CD8+ T-cells derived from peripheral blood or liver using major histocompatibility complex class I pentamers targeting immunodominant epitopes of HBV, Epstein-Barr-virus (EBV), or influenza virus (Flu). In chronic HBV infection, virus-specific CD8+ T-cells expressed higher levels of CD244 both in the peripheral blood and liver in comparison to the acute phase of infection or following resolution. CD244 was expressed at similarly high levels in EBV infection, but was low on Flu-specific CD8+ T-cells. In chronic HBV infection, high-level CD244 expression coincided with an increased expression of PD-1. The inhibition of the CD244 signaling pathway by antibodies directed against either CD244 or its ligand CD48 resulted in an increased virus-specific proliferation and cytotoxicity as measured by the expression of CD107a, interferon-γ, and tumor necrosis factor-α in CD8+ T-cells. CONCLUSION: CD244 and PD-1 are highly coexpressed on virus-specific CD8+ T-cells in chronic HBV infection and blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in chronic viral infections.

FOXP3 expression in hepatitis C virus-specific CD4+ T cells during acute hepatitis C.

BACKGROUND & AIMS: Down-regulation of hepatitis C virus (HCV)-specific CD4(+) T-cell responses is a hallmark of chronic viral persistence in acute hepatitis C. FOXP3(+)CD25(+)CD4(+) regulatory T cells can modulate HCV-specific immune responses in vitro, but the role of virus-specific regulatory T cells in the pathogenesis of chronic viral persistence is unknown. METHODS: Two novel HLA-DR15 tetramers were synthesized to study the kinetics and phenotype of FOXP3(+)-expressing HCV-specific CD4(+) T cells from 10 patients with acute hepatitis C and 15 patients with chronic hepatitis C. RESULTS: In acute hepatitis C, generally only a low percentage of HCV-specific CD4(+) T cells expressed FOXP3(+) (mean of 2.5% in patients with self-limited acute hepatitis C vs 2.4% in patients with evolving chronic hepatitis C). Although distinct but short-lived increases in virus-specific FOXP3(+)CD4(+) T cells occurred in 3 patients (30%, 26%, and 7% of tet(+) CD4(+) T cells, respectively), these did not correlate with the evolution of chronic hepatitis C. HCV-specific FOXP3(+)CD4(+) T cells displayed a distinct phenotype, with only 10% expressing CD25 and 40% being CD127low. Interestingly, this phenotype of FOXP3(+)CD4(+) T cells was already expanded in bulk CD4(+) T cells in patients with chronic hepatitis C. CONCLUSIONS: Although short-lived increases in HCV-specific FOXP3(+)CD4(+) T cells occur during the course of acute hepatitis C, we could not demonstrate an association of HCV-specific regulatory T cells and persistent viremia.

Antiviral therapy for recurrent hepatitis C after liver transplantation: sustained virologic response is related to genotype 2/3 and response at week 12.

OBJECTIVES: Recurrent hepatitis C virus (HCV) infection after liver transplantation (LT) is a major cause of transplant failure in HCV-positive patients. We retrospectively assessed the efficacy and safety of antiviral therapy and determined the factors influencing sustained virologic response (SVR) in LT recipients. METHODS: Between 1998 and 2007, we treated 36 LT recipients for hepatitis C cirrhosis and subsequent HCV recurrence (27 genotype 1 and 9 genotypes 2/3) with pegylated interferon alpha-2a (180 microg/week), pegylated interferon alpha-2b (1.5 microg/kg per week), or standard interferon alpha-2b (3 MIU 3X/week) plus ribavirin (600-1200 mg/day) for 48 weeks. RESULTS: SVR was achieved in seven of 27 (26%) of genotype 1 patients versus nine of nine (100%) genotype 2/3 patients (P=0.0001). Early virologic response at week 12 was associated with permanent viral clearance. Side effects included cytopenia and acute hearing loss, but rate of therapy withdrawal and dose reduction was low. CONCLUSION: Combination therapy in patients with HCV reinfection after LT yields an excellent SVR rate in genotype 2/3 patients, but remains unsatisfactory in genotype 1 patients. Virologic response at week 12 (early virologic response) can determine whether therapy should be continued or not.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection.

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Characterization of sequence variations in immunodominant regions of the HBV-nucleocapsid protein as a prerequisite for the development of an epitope-based vaccine.

BACKGROUND/AIMS: In hepatitis B virus infection, viral elimination is dependent on an efficient antiviral T cell response which is not detectable in chronic hepatitis B. Therefore, new therapeutic concepts focus on T cell activation, such as epitope-based T cell-targeted vaccines. However, with the development of peptide-based vaccines in mind, viral mutations frequently described in hepatitis B within known immunodominant helper epitopes may have an influence on peptide selection. METHODS: Mutant peptides within immunodominant epitopes (aa 1-20, aa 91-105, and aa 143-157) at position 12, 14, 93, 97, 147, 151, 153, and 155 were tested with peripheral blood mononuclear and specific clone cells for their ability to induce proliferation, produce cytokines, induce T cell receptor down-regulation or antagonize wild-type activity of the hepatitis B core antigen-specific CD4+ T cell clones. RESULTS: Five variants could not induce T cell proliferation or cytokine production when the variants were presented alone. Coincubation with wild-type epitopes leads to T cell activation showing that the variants do not act as T cell receptor antagonists for hepatitis B virus-specific CD4+ T cells. In contrast, five other variants and wild-type peptides stimulated CD4+ T cell proliferation and production of Th1 cytokines. CONCLUSIONS: Our data demonstrate that frequently occurring mutations within immunodominant epitopes have rather a nonstimulatory than a strengthening effect and thus should not included in a vaccine.

Leucocytapheresis with Adacolumn enhances HCV-specific proliferative responses in patients infected with hepatitis C virus genotype 1.

The most important aim in controlling virus infections is to destroy infected cells. Impaired cellular immunity in HIV and HCV infection leads to chronic infection. This study examined the effect of cytapheresis on the subsequent response to interferon/ribavirin treatment in patients infected with HCV. Adacolumn cytapheresis was carried out once a day for 5 consecutive days in patients who relapsed or did not respond to previous peginterferon and ribavirin combination treatment (n = 14: relapsers = 3, non-responders = 11). Peginterferon and ribavirin combination treatment was started after cytapheresis. During combination treatment, the proliferative response of peripheral blood mononuclear cells to HCV proteins (core, NS3, NS4, and NS5), tetanus toxoid, and phytohemagglutinin was measured, and compared to the early virological response. After treatment by leucocytapheresis, the proliferative response of peripheral blood mononuclear cells to HCV-core and tetanus toxoid increased significantly over the baseline (P < 0.05). A marked increase in the phytohemagglutinin response was observed after peginterferon and ribavirin combination treatment was started (P < 0.01 at week 5 and P < 0.005 at week 13). There were, however, no clear changes in the proliferative response to other antigens. Among the 14 patients, 12 (85.7%) achieved an early virological response by week 13 (12 weeks after the start of combination treatment). After treatment, nine patients (64.3%) had a significant proliferative response to HCV core antigen. Among the nine patients, eight patients (88.9%) achieved early virological response. The results indicate that activation of cellular immunity by leucocytapheresis facilitates an early virological response rate in HCV patients. This new therapy may, therefore, become an additional therapeutic measure for HCV.

Plasmacytoid dendritic cells in acute and chronic hepatitis C virus infection.

Chronic evolution of acute hepatitis C (aHC) occurs in more than 80% of patients but can frequently be prevented by early treatment with interferon (IFN)-alpha. Plasmacytoid dendritic cells (pDCs) are the major endogenous IFN-alpha producers, but their role in aHC is unknown. In this study, frequency, phenotype, and pDC function were analyzed in 13 patients with aHC and 32 patients with chronic hepatitis C (cHC) compared with 20 healthy controls, 33 sustained responders to antiviral treatment, 14 patients with acute hepatitis B (aHB), and 21 patients with nonviral inflammatory disease. In aHC, pDCs in the peripheral blood were significantly reduced compared with healthy controls (median, 0.1% vs. 0.36%, P < .0005) and were inversely correlated to alanine aminotransferase levels (r = -0.823; P < .005). Circulating pDCs in aHC were immature, as determined via reduced expression of HLA-DR and CCR7, and produced little amounts of IFN-alpha (median, 3.5 pg/50,000 peripheral blood mononuclear cells [PBMCs] vs. 498.4 pg/50,000 PBMCs in healthy controls; P < .0005). Less pronounced changes were present in cHC (median, 0.17%, 28.0 pg/50,000 PBMCs IFN-alpha, respectively). However, a significantly reduced frequency and IFN-alpha production was also found in self-limited aHB (median 0.1%, 8.6 pg/50,000 PBMCs) and in patients with nonviral inflammatory disease (median 0.19%, 7.5 pg/50,000 PBMCs). In conclusion, in aHC frequency and IFN-alpha-producing capacity of peripheral blood pDCs are dramatically reduced and inversely correlated with the degree of liver inflammation. In cHC there is incomplete recovery of pDC function, which, however, could be solely due to the chronic inflammatory state.

Uptake and presentation of hepatitis C virus-like particles by human dendritic cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Interaction of dendritic cells (DCs) with viral particles may play an important role in the immunopathogenesis of HCV infection. Since the synthesis or purification of infectious virions is limited, we used HCV-like particles (HCV-LPs) to study the interaction of HCV with human DCs. Immature DCs exhibited an envelope-specific and saturable binding of HCV-LPs, indicating receptor-mediated DC-HCV-LP interaction. Confocal microscopy revealed that HCV-LPs were rapidly taken up by DCs in a temperature-dependent manner. Competition experiments demonstrated that C-type lectins such as mannose receptor or DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing nonintegrin) were not sufficient for mediating HCV-LP binding. HCV-LP uptake was followed by DC activation. DCs pulsed with HCV-LPs stimulated HCV core-specific CD4(+) T cells, indicating that uptake of HCV-LPs by DCs leads to antigen processing and presentation on major histocompatibility complex (MHC) class II molecules. Finally, HCV-LP-derived antigens were efficiently cross-presented to HCV core-specific CD8(+) T cells. These findings demonstrate that HCV-LPs represent a novel model system to study HCV-DC interaction allowing definition of the molecular mechanisms of HCV uptake, DC activation, and antigen presentation to T cells. Furthermore, HCV-LP-mediated DC activation and efficient antigen presentation may explain the marked immunogenicity of HCV-LPs in vivo.

Analysis of a successful HCV-specific CD8+ T cell response in patients with recurrent HCV-infection after orthotopic liver transplantation.

Virus-specific CD8+ T cells play a major role in antiviral immune defenses; their significance in the transplant setting, however, is unclear. In the present study, we asked whether hepatitis C virus (HCV)-specific CD8+ T cells were detectable in the presence of an immunosuppressive treatment and whether the HCV-specific CD8+ T cell response correlates with treatment outcome in patients who receive interferon (IFN)-alpha / ribavirin therapy after orthotopic liver transplantation (OLTx). Liver- and blood-derived T cell lines of 21 patients after OLTx were studied before, at the end of, and after antiviral treatment. Virus-specific IFN-gamma production in response to a panel of previously identified HCV-specific epitopes restricted by the human leukocyte antigen (HLA) class I molecules A2, A3, B7, B35, and B44 of structural and nonstructural HCV protein was determined by enzyme-linked immunospot (ELISPOT) assay. Before treatment, only low numbers of HCV-specific CD8+ T cells were detectable. In 6 patients with a sustained virological response, a significant, multispecific, and sustained CD8+ T cell response was detectable, which was mainly found in the peripheral blood. Nonresponders and transient responders showed undetectable, weak, or transient HCV-specific CD8+ T cell responses. (Sustained responders vs. transient and nonresponders: Wilcoxon rank-signed test; P < .01). In conclusion, our data indicate that despite immunosuppression, HCV-specific CD8+ T cells are detectable in patients with recurrent HCV infection after OLTx and that a significant, multispecific, and long-lasting HCV-specific CD8+ T cell response contributes to viral elimination.

Antiviral treatment of recurrent hepatitis C virus (HCV) infection after liver transplantation: association of a strong, multispecific, and long-lasting CD4+ T cell response with HCV-elimination.

BACKGROUND/AIMS: Patients with recurrent hepatitis C virus (HCV)-infection after liver transplantation (OLTx) could develop an early, multispecific, preferentially intrahepatic CD4+ T cell response. We asked now whether there is a correlation between the HCV-specific CD4+ T cell response and treatment outcome in patients who receive interferon (IFN)-alpha/ribavirin. METHODS: Liver- and blood-derived T cell lines of 20 patients were studied in parallel before, under, at the end and after antiviral treatment. Virus-specific IFN-gamma production at a single cell level to HCV-proteins (core, non-structural protein (NS)3/4, NS5) was determined by enzyme-linked immunospot assay. RESULTS: In 6/7 non-responders a weak HCV-specific CD4+ T cell response was detectable. All six sustained responders developed a strong, at NS3/4 and NS5 directed and long-lasting CD4+ T cell response which was mainly detected in peripheral blood mononuclear cells. This reaction was significantly stronger: (1) in the responders than in the non-responders; and (2) within the responders at the end of treatment than before (P<0.03). Seven transient-responders showed a weak and/or transient HCV-specific CD4+ T cell response. CONCLUSIONS: In patients with recurrent HCV-infection after OLTx, who receive antiviral treatment, a strong, at NS3/4 and NS5 directed and long-lasting CD4+ T cell response is associated with HCV-elimination whereas no or a weak/transient response is associated with treatment failure.

Acute hepatitis C: high rate of both spontaneous and treatment-induced viral clearance.

BACKGROUND & AIMS: Acute hepatitis C virus infection accounts for approximately 20% of cases of acute hepatitis today. The aim of this study was to define the natural course of the disease and to contribute to the development of treatment strategies for acute hepatitis C virus. METHODS: The diagnosis of acute hepatitis C virus in 60 patients was based on seroconversion to anti-hepatitis C virus antibodies or clinical and biochemical criteria and on the presence of hepatitis C virus RNA in the first serum sample. RESULTS: Fifty-one of 60 (85%) patients presented with symptomatic acute hepatitis C virus. In the natural (untreated) course of acute symptomatic hepatitis C (n = 46), spontaneous clearance was observed in 24 patients (52%), usually within 12 weeks after the onset of symptoms, whereas all asymptomatic patients (n = 9) developed chronic hepatitis C. The start of antiviral therapy (interferon-alpha with or without ribavirin) beyond 3 months after the onset of acute hepatitis induced sustained viral clearance in 80% of treated patients. CONCLUSIONS: The management of acute hepatitis C has to take into account the high rate of spontaneous viral clearance within 12 weeks after the onset of symptomatic disease. Treatment of only those patients who remain hepatitis C virus RNA positive for more than 3 months after the onset of disease led to an overall viral clearance (self-limited and treatment induced) in 91% of patients, and unnecessary treatment was avoided in those with spontaneous viral clearance. Patients with asymptomatic acute hepatitis C virus infection are unlikely to clear the infection spontaneously and should be treated as early as possible.

Upregulation of major histocompatibility complex class I on liver cells by hepatitis C virus core protein via p53 and TAP1 impairs natural killer cell cytotoxicity.

The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis C virus (HCV) are still unclear. Here, we present evidence for a cascade of molecular events that the virus initiates to subvert the innate immune attack. The HCV core protein induced p53-dependent gene expression of TAP1 (transporter associated with antigen processing 1) and consecutive major histocompatibility complex (MHC) class I upregulation. Moreover, in p53-deficient liver cell lines, only reconstitution with wild-type p53, but not mutated p53 lacking DNA binding capacity, showed this effect. As a consequence of increased MHC class I expression, a significantly downregulated cytotoxic activity of natural killer (NK) cells against HCV core-transfected liver cells was observed, whereas lysis by HCV-specific cytotoxic T cells was not affected. These results demonstrate a way in which HCV avoids recognition by NK cells that may contribute to the establishment of a chronic infection.

Detection of functionally altered hepatitis C virus-specific CD4 T cells in acute and chronic hepatitis C.

Chronic hepatitis C is characterized by a weak or absent hepatitis C virus (HCV)-specific CD4(+) T-cell response in terms of antigen-specific proliferation or interferon gamma (IFN-gamma) secretion. To clarify whether this is due to the absence or functional impairment of antigen-specific CD4(+) T cells we developed an assay that relies on the induced expression of the T-cell activation marker CD25 and is therefore independent from cytokine secretion or proliferation. In 10 of 20 patients with chronic hepatitis C, a significant number of antigen-specific activated CD4(+) T cells (mean 1.06%/patient; range, 0% to 5.2% of CD4(+) T cells) could be shown, whereas antigen-specific proliferation was present in only 1 of 20 patients. IFN-gamma secretion was absent in all 13 patients tested. However, significant antigen-specific interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) secretion was present in 6 of 10 and 3 of 10 patients, respectively. In 8 patients with acute hepatitis C, irrespective of disease outcome, HCV-specific CD4(+) T cells were detected in all patients and at a significantly higher frequency (mean 3.7%/patient; range, 1.16% to 7.17%) in the first weeks of disease. A chronic course of disease was associated either with a loss of both IFN-gamma secretion and proliferation, resembling an anergic state, or a loss of T-cell proliferation followed by a rapid decline in IFN-gamma-producing cells, corresponding to exhaustion of the specific immune response. In conclusion, functional changes of HCV-specific CD4(+) T cells or failure to develop a long-lasting T-helper response may contribute to chronic hepatitis C viral persistence.

Hepatitis C virus eradication associated with hepatitis B virus superinfection and development of a hepatitis B virus specific T cell response.

BACKGROUND/AIMS: Specific T cell responses during acute hepatitis B and during chronic hepatitis C have been described in detail. However, the T cell responses during the rare setting of acute hepatitis B virus (HBV) infection in the course of chronic hepatitis C that eventually lead to clearance of both viruses are completely unknown. METHODS: We analyzed the virus specific CD4+ and CD8+ T cell response during an acute HBV superinfection in a patient with chronic hepatitis C. RESULTS: The patient eliminated hepatitis C virus (HCV)-RNA and HBV-DNA from serum soon after the clinical onset of acute hepatitis B. The HBV specific T cell response found in this patient corresponds to the typical response that has been described in acute hepatitis B without chronic HCV infection. In contrast the hepatitis C specific immune response was similar to that generally found in chronic hepatitis C despite the fact that the patient also eliminated HCV-RNA. CONCLUSIONS: We hypothesize that the acute HBV infection induced a HBV specific T cell response which was associated with elimination HBV DNA and HCV-RNA, the latter possibly by bystander mechanisms, e.g. via secretion of cytokines. If such a non-specific bystander mechanism which has proven to be effective in the experimental setting and which is formally described here for a single patient can be shown to be a more general phenomenon, it may support the approach with new antiviral strategies, e.g. the induction of non-specific defense mechanisms against HCV.